Bacteria of the genus Neisseria are responsible for causing two relatively common human diseases. Neisseria gonorrhoeae is the causative agent for gonorrhea and Neisseria meningitidis is the pathogen for one type of bacterial meningitis. Obviously, it is important for laboratories and physicians to be able to identify these bacteria quickly and accurately.
The use of chromogenic substrates to detect preformed enzymes for the identification of pathogenic Neisseria species was first described by D'Amato et al., J. Clin. Microbiol., 7, 77-81 (1978).
Commercial products utilizing enzymatic substrates have generally been known to be simple, accurate, and relatively rapid. See for example, Janda, et al., J. Clin. Microbiol., 21, 734-737 (1985); Philip et al., J. Clin. Microbiol., 22, 101-104 (1985) and Welborn, et al., J. Clin. Microbiol., 20, 680-683 (1984).
Most of these systems rely on the solution phase detection of three preformed enzymes, prolylamino peptidase (PAP), gamma-glutamylaminopeptidase (GAP), and Beta-D-galactosidase (BDG), which are associated with Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica, respectively.
It has also been reported that the solution phase detection of these enzymes approaches 100% specificity for each of the three Neisseria species. See, D'Amato, et al., supra; Yajko, et al., J. Clin. Microbiol., 19, 380-383 (1984); Janda, et al., supra; Philip et al., supra; and Welborn, et al., supra.
One commercially available test for the identification of Neisseria species from isolation media is Gonochek II.RTM. (E. I. Du Pont de Nemours & Co., Wilmington, DE 19898). The Gonochek II test utilizes three chromogenic substrates contained in a single tube to detect preformed enzymes associated with the three Neisseria species.
Suspect organisms from suitable selective media are transferred to the Gonochek tube and incubated for 30 minutes at 35.degree. C. After the addition of reagent, color reactions reveal specific enzymes in the solution. These enzymes are confirmatory for three Neisseria species; prolylaminopeptidase for N. gonorrhoeae, gamma-glutamylaminopeptidase for N. meningitidis, and beta-d-galactosidase for N. lactamica.
When an isolate is removed from selective media, detection of PAP alone identifies Neisseria gonorrhoeae, GAP identifies Neisseria meningitidis, and BDG identifies Neisseria lactamica. However, exceptions have been noted. For example, using the Gonocheck II system, Philip et al., supra, found that 4.6% of the Neisseria meningitidis strains were negative for GAP, while Janda et al., supra, found 5 of 172 (3%) of the Neisseria meningitidis isolates negative for GAP. PAP positive Neisseria meningitidis isolates have also been reported by Yajko et al., supra, who found 43% positive and by D'Amato et al., supra, who reported 6.7% positive.
The present invention is directed to a system (i.e., both a product and process) designed to identify these same enzymes from Neisseria species on a solid absorbent, e.g., filter paper, rather than in solution.
The preferred test strip of the present invention comprises three sections of filter paper each impregnated with one of the three Neisseria species specific substrates. After the addition of a buffer, the suspect organism is smeared on each filter paper section, and the strip is incubated at 35.degree. C. for 10 minutes at 35.degree.-37.degree. C. or 20 minutes at room temperature. If no color develops in section A after the incubation period, a color developing reagent is added to produce a color reaction on selected sections.
In comparison to the solution phase assay methods of the prior art, the assay test of the present invention has been found to be:
1. more rapid (immediate color formation after incubation) PA1 2. more sensitive (intense color formation) PA1 3. less subjective (clear results +/-) PA1 4. more convenient (no messy tubes, etc.)
There is no other commercially available direct enzymatic test system designed to detect all three Neisseria species on filter paper. Additionally, although the color reagent preferably employed (dimethylaminocinnamin-aldehyde) has been previously used in solution by microbiologists (the API system is one example), never before has it been adapted for use in a commercial test on filter paper for the rapid identification of bacterial polyaminopeptidase and gamma-glutamy/aminopeptidase.